Biochemistry Laboratory – CH600 (2008-2009) Experiment 9

*Michelle Dy Sim, Gellina Ann Ram Suderio, Jonnah Kristina Chua Teope Department of Biology, 3Biology-6, Group # 7, College of Science University of Santo Tomas, España Street, Manila 1008
March 2, 2009 Abstract: Urine is a liquid waste product of the body secreted by the kidneys by a process of filtration from blood called urination and excreted through the urethra. Urinalysis is an array of tests performed on urine and one of the most common methods of medical diagnosis. The objectives of this experiment are to subject the urine sample acquired to several tests and to qualitatively examine the presence of some normal organic constituents and pathologic organic constituents. Initial examination includes the notation of the collection time, color, turbidity, acidity and pH of the urine sample. For the qualitative exam of normal organic constituents, the test for urea, uric acid, indican and creatinine were conducted. In these tests, the patient’s results were all normal. For the qualitative examination for pathologic organic constituents, Gunnings’s test, Benedict’s test, Exton’s test, Smith’s test and Occult blood test were performed. The result obtained for the urine sample was that the patient was healthy except for the fact that the patient might have diabetes. Keywords: • Urine • Urinalysis • Laboratory Tests

I. Introduction Urine is a transparent solution that can range from colorless to amber but is usually pale yellow. Urine is an aqueous solution of metabolic wastes such as urea, dissolved salts, and organic compounds produced by the kidneys. It plays a vital role in maintaining homeostasis. The production of urine is called diuresis.[1] Urine ranges from pale yellow to amber because of the pigment urochrome. The color indicates the concentration of the urine and varies with specific gravity. Dilute urine is straw colored, while concentrated urine is deep amber.[2]

The aromatic odor of fresh, normal urine is caused by the presence of volatile acids. Analysis of the pH of a freshly voided urine specimen indicates the acid – base balance. The urine reflects the work of the kidneys to maintain normal pH homeostasis.[2] The kidneys maintain normal acid – base balance primarily through reabsorption of sodium and tubular secretion of hydrogen and ammonium ions.[3] Urinalysis is part of routine diagnostic and screening evaluations performed on urine that provide a general overview of a person’s health.[4] Urinalysis is used as a diagnostic tool because it can help detect substances or cellular material in the urine associated with different metabolic and kidney disorders. It is routinely done in all patients admitted to the hospital, pregnant women and presurgical patients. It is done diagnostically in patients with abdominal or back pain, dysuria, hematuria, or urinary frequency. It is part of routine monitoring in patients with chronic renal disease and some metabolic diseases.[2] Most urine tests are performed for one of the following reasons: to diagnose renal or urinary tract disease; to monitor renal or urinary tract disease; to detect metabolic or systematic diseases not directly related to the kidneys.[2] The process of urinalysis determines the following properties of urine: color, odor, turbidity, specific gravity, pH, glucose, ketones, blood, protein, bilirubin, urobilinogen, nitrite, leukocyte estaerase, and other abnormal constituents revealed by microscopic examination of the urine sediment.[3] This experiment aims to subject the urine sample acquired to several tests and to qualitatively examine the presence of some normal organic constituents and pathologic organic constituents.

II. Methodology A. Initial Examination of Urine The color, turbidity, acidity and the time when the urine was collected was noted.

B. Qualitative Examination for Normal Organic Constituents 1.) Test for Urea A 0.5mL of 70% NaOH and 4 drops of bromine water was added to 1mL urine sample. After, the evolution of N2 gas was observed. 2.) Test for Uric Acid A 1mL of urine sample was added with 5mL of 20% Na2CO3. Then, 5 drops of phosphotungstic acid reagent was also added then mixed. The formation of a blue color was observed. 3.) Indican Test To begin with, 5mL of Obemayer’s reagent was added to 5mL urine sample then mixed. Then, 3mL of chloroform was added to it before shaking and allowed to settle. The formation of a blue color in the lower chloroform layer was observed. 4.) Test for Creatinine A 1mL alkaline picrate solution was added to 2mL urine sample. The formation of an orange – colored solution was noted. C. Qualitative Examination for Pathogenic Organic Constituents 1.) Gunning’s Test The 5mL of urine sample was basified with 5 drops of concentrated ammonium hydroxide using a red litmus paper. Then, Lugol’s solution was added to the basic urine, enough to produce a black cloud which does not disappear immediately. It was let stood for 5 minutes. Similar procedure was done for the positive control. The results obtained were compared. 2.) Benedict’s Test To start with, 5mL Benedict’s reagent was added to 8 – 10 drops of urine sample in a test tube then mixed. It was then heated in a boiling water bath for 2 – 3 minutes then let stood and allowed to cool. The formation of a precipitate was observed. Similar procedure was done for the positive control. The results obtained were compared.

3.) Exton’s Test First, 3mL of urine sample and 3mL of Exton’s reagent were mixed in a test tube. The solution was warmed until cloudiness appeared. Similar procedure was done for the positive control. The results obtained were compared. 4.) Smith’s Test The 5mL urine sample was placed in a test tube. The test tube was inclined and overlayed with 3mL tincture of alcoholic iodine mixture. Similar procedure was done for the positive control. The results obtained were compared. 5.) Test for Occult Blood A half spatula guaiac powder was added with 5mL of 95% ethanol in a test tube then mixed. To this mixture, 5mL hydrogen peroxide was added. Then, 5mL of this solution was added to 3mL acidified urine. Similar procedure was done for the positive control. The results obtained were compared.

III. Results and Discussion Urea is the major end product of protein nitrogen metabolism in humans. Hepatic enzymes convert ammonia from amino acids to urea.[5] Urea is produced in the liver and excreted through the kidneys in the urine. The circulating levels of urea depend upon protein intake, protein catabolism and kidney function. Elevated urea levels can occur with dietary changes, diseases which impair kidney function, liver diseases, congestive heart failure, diabetes and infections. A positive result shows the formation of N2 gas or moistening of the sides of the test tubes used. The urea cycle (also known as the ornithine cycle) is a cycle of biochemical reactions occurring in many animals that produces urea (NH2)2CO from ammonia (NH3). This cycle was the first metabolic cycle discovered. In mammals, the urea cycle takes place only in the liver.[6]

Uric acid is an organic compound of carbon, nitrogen, oxygen and hydrogen with the formula C5H4N4O3. Xanthine oxidase oxidizes oxypurines such as xanthine and hypoxanthine to uric acid. Uric acid is the final breakdown product of purine catabolism.[2] In most other mammals, the enzyme uricase further oxidizes uric acid to allantoin. Uric acid is a strong reducing agent and a potent antioxidant. In humans, about half the oxidant capacity of plasma comes from uric acid. Humans produce large quantities of uric acid. In human blood, uric acid concentrations between 3.6mg/dL and 8.3mg/dL are considered normal by the American Medical Association, although significantly lower levels are common in vegetarians due to a decreased intake of purine – rich meat.[2] Diseases related to elevated uric acid levels are kidney stones, Lesch – Nyhan syndrome, CVDs and diabetes. Uric acid reacts with phosphotungstic acid to produce allantoin and tungsten blue.


H3P W12O40

+ Tungsten Blue

Indican is an indole produced by bacterial action on an amino acid, tryptophan, in the intestine. Most of the indole is excreted in the feces. The remainder is absorbed, metabolized and excreted as indicant in the urine. In normal urine, the amount of indican excreted is small. It is increased with high protein diets or inefficient protein digestion. If not digested properly, or if the wrong types of proteins are ingested, bowel putrefaction can occur. Problems with protein digestion can be caused by overgrowth of anaerobic bacteria, intestinal obstruction, stomach cancer, low stomach acid, parasitic infections, malabsorptive syndromes, fungal infections, lack of digestive enzymes, or liver problems. Following absorption, indole is converted to 3-hydroxy indole in the liver. Detection of indican in the urine depends upon its decomposition and subsequent oxidation of indoxyl to indigo blue and its absorption into a chloroform layer.

+ HO 
2 2

+ Indoxyl Sulfuric Acid K

Creatinine test measures the level of the waste product creatinine in the urine. This test indicates whether the kidneys are working properly. Creatine is formed when food is changed into energy through a process called metabolism. Creatine is broken down into another substance called creatinine by the

addition of strong acid or by alkali or by using enzyme, creatine hydroxylase.[7] If the kidneys are damaged and cannot work normally, the amount of creatinine in the urine goes down while its level in the blood goes up. Alkaline picrate solution is composed of saturated picric acid and 10% NaOH. Most methods used for creatinine determination are based upon the Jaffe reaction. The Jaffe reaction uses saturated picric acid which oxidizes creatinine in alkali forming creatinine picrate, in which creatinine forms a characteristic orange color when treated with alkaline picrate.[7]

Gunning’s test checks for ketone bodies in the blood or urine. Ketone bodies are three water – soluble compounds that are produced as by – products when fatty acids are broken down for energy in the body. The excess presence of ketones in urine is associated with diabetes or altered carbohydrate metabolism.[3] Lugol’s solution consists of 5% iodine and 10% potassium iodide in 85% distilled water with a total iodine content of 130mg/mL. A positive result shows a formation of iodoform crystals. Glucose levels are measured to diagnose diabetes. The normal glucose level in the urine is 180mg/dL. Urine glucose levels of 300 – 500mg/dL are common with severe untreated diabetes. Diabetes results from deficient insulin or decreased sensitivity to insulin. The results of a urine glucose test are abnormal in cases of renal glycosuria and diabetes mellitus.[2]

+ 2Cu


+ 4OH-

 Cu O


+ 2H2O

Normally, protein is not found in urine. This is because the kidney is supposed to keep large molecules in the blood and only filter out smaller impurities. If the kidney is diseased, protein will appear in the urine. Exton’s reagent is 5% sulfosalicylic acid in a solution of sodium sulfate. A cloudy solution shows the presence of albumin. Hemoglobin breakdown results in bilirubin production. In the liver, bilirubin is conjugated to an acid to make conjugated bilirubin. Unconjugated bilirubin is water soluble and can be excreted in the

urine. Abnormal bilirubin values may indicate anemia, excessive breakdown of RBC, hepatitis, cirrhosis, obstruction of biliary duct, toxic liver damage and biliary tree obstruction.[3] Tincture of alcoholic iodine is usually 10% elemental iodine in ethanol. Addtion od a solution of alcoholic iodine to urine produces a green color. The green color indicates the presence of bile. The addition of hydrogen peroxide to 5mL 95% ethanol and guaiac powder oxidizes the guaiac causing a color change. Heme, a component of hemoglobin found in blood, catalyzes this reaction giving a result in about two seconds. A blue ring indicates a positive result.[8] A. Initial Examination of Urine Sample Time collected Color Turbidity Acidity pH Early morning Light Clear Neutral 7

B. Qualitative Exam of Normal Organic Constituents Test UREA URIC ACID INDICAN CREATININE Result With little evolution of white fumes; red litmus paper Formation of blue – colored solution Formation of blue – colored interface in the lower chloroform layer Yellow to orange – colored solution

C. Qualitative Exam for Pathologic Constituents Test GUNNING’S TEST (ketone bodies) BENEDICT’S TEST (glucose) EXTON’S TEST (albumin) SMITH’S TEST (bile pigments) OCCULT BLOOD TEST Positive (+) Control Cloudy interface with crystals Olive green cloudy solution with precipitate Cloudy solution Emerald green – colored solution with no precipitates X Urine Sample Light orange solution with red precipitate Clear green solution with some cloudy precipitate No cloudiness Yellow orange interface; no emerald green color X

A normal urine specimen should be clear. Cloudy urine may be caused by the appearance of pus, RBCs or bacterias; however, normal urine also may be cloudy because of ingestion of certain foods. Abnormally colored urine may also result from a pathologic condition or the ingestion of certain medicines. Dark yellow urine may indicate the presence of urobilinogen or bilirubin.[2]

The kidneys assist in acid – base balance by reabsorbing sodium and excreting hydrogen. An alkaline pH is observed in a patient with alkalemia. Bacteria, urinary tract infection, or a diet in citrus fruits or vegetables may cause increased urine pH. Alkaline urine is associated with calcium carbonate, calcium phosphate and magnesium phosphate stones. Acidic urine is generally obtained from patients with academia, which can result from metabolic or respiratory acidosis, starvation, dehydration, or a diet high in meat products or cranberries. Acidic urine is associated with xanthine, cystine, uric acid and calcium oxalate stones.[2] Urine pH becomes alkaline on standing, because of the action of urea – splitting bacteria, which produce ammonia. The urine pH of an uncovered specimen will become alkaline because carbon dioxide vaporizes from the urine. Dietary factors affect urine pH. Ingestion of large quantities of citrus fruits, dairy products, and vegetables produces alkaline urine, whereas a diet high in meat and certain foods produces acidic urine.[2] Protein is a sensitive indicator of kidney function. Normally, protein is not present in the urine because the spaces in the normal glomerular filtrate membrane are too small to allow its passage. If glomerular membrane is injured, the spaces in the filtrate become larger, and the protein seeps into the filtrate, then into the urine. If this persists at a significant rate, hypoproteinemia can develop as a result of severe protein loss through the kidneys. This decreases the normal capillary oncotic pressure that holds fluid within the vasculature and causes severe interstitial edema.[2] Specific gravity is a measurement of the kidney’s ability to concentrate urine.[3] Specific gravity is used to evaluate the concentrating and excretory power of the kidneys. High specific gravity indicates concentrated urine. Low specific gravity indicates dilute urine. Specific gravity refers to the weight of the urine compared with that of distilled water. Particles in the urine give it weight or specific gravity.[4] Leukocyte (WBC) esterase is a screening test used to detect leukocytes in the urine. Positive results indicate urinary tract infection. Leukocyte esterase is nearly 90% accurate in detecting WBC in urine.[2]

Like the leukocyte esterase screen, the nitrite test is a screening test for identification of urinary tract infection. Nitrite screening enhances the sensitivity of the leukocyte esterase test to detect urinary tract infection. Nitrite testing is only about 50% accurate in detecting WBCs in the urine.[2] Normally, no ketones are present in the urine; however, a patient with poorly controlled diabetes and hyperglycemia may have a massive fatty acid catabolism. The purpose of this catabolism is to provide an energy source when glucose cannot be transferred into the cell because of insulin insufficiency. Ketones are the end products of this fatty acid breakdown.[2] Bilirubin is a major constituent of bile. If bilirubin excretion is inhibited, conjugated hyperbilirubinemia will result. Bilirubin in urine suggests disease affecting bilirubin metabolism after conjugation or defects in excretion. For screening, elevated urine bilirubin concentration can indicate previously unsuspected liver injury due to disease, gallstones or drug toxicity. Bilirubin in the urine will color the urine dark yellow or orange.[2]

IV. Conclusion It comes to the conclusion that the patient is therefore free form any sickness aside from the fact that the patient is already close to having diabetes, and should now be watchful of her sugar level intake.

V. References [1] Urinalysis – Retrieved February 27, 2009 http://en.wikipedia.org/wiki/Urinalysis [2] Pagana, K. D. and Pagana, T. J. Mosby’s Manual of Diagnostic and Laboratory Tests. St. Louis: Mosby, 2002. [3] Fischbach, F. and Dunning, M. B. III. A Manual of Laboratory and Diagnostic Tests. Philadelphia: Williams & Wilkins, 2004. [4] Urea Cycle – Retrieved February 28, 2009 http://en.wikipedia.org/wiki/UreaCycle [5] Harr, R. R. Clinical Laboratory Science Review. Philadelphia: F.A. Davis, 2007. [6] Wallach, J. Interpretation of Diagnostic Tests. Philadelphia: Wolters Kluwer Health/Lippincott Wiliams & Wilkins, 2007. [7] Urine – Retrieved February 27, 2009 http://en.wikipedia.org/wiki/Urine [8] Lehmann, C. A. Saunder’s Manual of Clinical Laboratory Science. Philadelphia: W.B. Saunders, 1998. [9] Worthley, L. I. G. Handbook of Emergency Laboratory Tests. New York: Churchill Livingstone, 1996.

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